Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education; Beijing, , People's Republic of China

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1 Concise Communication DOI /art Variants in CCR6 are associated with susceptibility to lupus nephritis in Chinese Authors: Xu-jie Zhou 1, MD & PhD;Rong Mu 2, MD & PhD;Chun Li 2, MD;Swapan K Nath 3, PhD;Yue-miao Zhang 1, MD;Yuan-yuan Qi 1, MD;Zhan-guo Li 1, MD & PhD;Ming-hui Zhao 1, MD & PhD;Hong Zhang 1, MD & PhD Author affiliations: 1, Renal Division, Peking University First Hospital; Peking University Institute of Nephrology; Key Laboratory of Renal Disease, Ministry of Health of China; and Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education; Beijing, , People's Republic of China 2, Department of Rheumatology and Immunology, Peking University People's Hospital, Beijing, , China. 3, Arthritis and Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Correspondence to: Prof. Hong Zhang, MD, PhD Renal Division, Peking University First Hospital, Peking University Institute of Nephrology No.8 Xi Shi Ku Street, Xi Cheng District, Beijing (China) Tel: , Fax: , hongzh@bjmu.edu.cn Funding: This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an Accepted Article, doi: /art American College of Rheumatology Received: Jan 15, 2015; Revised: May 18, 2015; Accepted: Jun 25, 2015

2 Page 2 of 9 This work was supported by grants from the Major State Basic Research Development Program of China (973 program, No. 2012CB517700), the National Natural Science Foundation of China (No ), Beijing Natural Science Foundation(No and ), the Research Fund of Beijing Municipal Science and Technology for the Outstanding PhD Program ( ), The Foundation of Ministry of Education of China ( ), the Natural Science Fund of China to the Innovation Research Group ( ) and the NIH (AR and AI107176). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure of potential conflict of interest: None declared.

3 Page 3 of 9 Systemic lupus erythematosus (SLE) is a common autoimmune disease with a complex genetic etiology. In the past two years, exciting insights supporting the proinflammatory activity of IL-17-producing T-helper 17 subset (Th17 cells) in the pathogenesis of several autoimmune or allergic diseases, including asthma, atopic dermatitis, rheumatoid arthritis (RA) and SLE, have emerged(1,2). High production of IL-17 is observed in SLE patients and several compounds targeting IL-17 have demonstrated marked clinical efficacy (1,2). Genetic studies have reproducibly demonstrated an association between susceptibility to RA and polymorphisms of the CCR6 gene, a surface marker for Th17 cells (3,4). However, to date, no direct link between CCR6 and SLE, has been found from genetic studies, although higher percentages of CCR6+ T helper cells were observed in patients with SLE especially in lupus nephritis (LN) compared to controls (5,6). Thus the current study sought to investigate whether there are any associations between CCR6 polymorphisms and susceptibility to LN. In the current study, discovery cohort comprised of 500 LN patients (age 32.0±11.5 years, female 83.6%) and 900 unrelated healthy individuals (31.5±8.4 years, female 40.1%). And replication cohort comprised of 626 LN patients (32.5±12.8 years, female 84.0%) and 1932 healthy individuals (40.9±12.7 years, female 47.7%) as well as 1063 SLE patients without indicators of renal involvement (36.6±13.4 years, female 90.5%) as controls. All the patients met the revised SLE criteria of the American College of Rheumatology(7). The study was approved by the medical ethics committee of Peking University. All patients gave informed consent. Two intronic SNPs rs and rs known to be associated with RA with top association signals were selected(3, 4) and genotyping was undertaken by TaqMan allele discrimination assays (Applied Biosystems, FosterCity, California, USA) as previously reported(8, 9). Direct sequencing was performed in randomly selected 45 samples on the basis of rs genotype (15 subjects with A/A, 15 with G/A, and 15 with G/G genotype) to determine whether rs could tag the recently identified functional CCR6DNP (a triallelic dinucleotide polymorphism of CCR6) (3). Power of the study was calculated by CaTS

4 Page 4 of 9 ( As rs and rs are in high linkage disequilibrium ( r ), indicating statistical tests performed on each SNP are actually highly dependent, no multiple correction was applied. Statistical analyses were performed with SPSS16.0 software (SPSS Inc., Chicago, IL). Functional annotations of variants were obtained from HaploReg and regulomedb databases. A total 5021 Chinese were included and the call rate for rs and rs were 99.82% and 98.94%. Both SNPs studied were in Hardy-Weinberg equilibrium in controls and patients (p>0.05). With the expected frequency of rs and rs minor allele (40%), we had a power of 96-99% to detect a 1.20 fold increased risk at separated stages and in joint. The frequency of the A allele of rs was significantly higher in LN both in discovery cohort and replication cohort as compared with healthy controls (Table 1, p= and p = , respectively). This difference became more significant for the combined groups (p= , OR 1.18, 95% CI ). Comparing with patients of SLE without LN, increased frequencies of rs a and rs a were also observed in LN patients (Table 2, p<0.05). Dominant models showed better fit whereas additive models also showed significant associations between CCR6 variants and SLE (Table 2), consistent with the data from rheumatoid arthritis. And logistic regression analysis adjusted by sexes and ages also suggested that risk genotypes of rs (AA+AG, p= 0.02, OR 1.21, 95% CI ) and rs (AA+AG, p= 0.01, OR 1.24, 95% CI ) were associated with LN compared to controls. As the two SNPs were in high LD, risk haplotype AA also showed significant associations with LN compared to controls (p= , OR 1.08, 95% CI ). However, no other phenotypic (serum creatinine, complement level, SLEDAI, proteinuria, and type of LN) correlations were observed (p>0.05). By searching ENCODE data, the rs was observed to locate in region of promoter histone marks in GM12878 (B lymphoblastoid) and rs is a cis-expression SNP for CCR6 in monocyte. In addition, direct sequencing of the functional dinecleotide polymorphism of CCR6, CCR6DNP, revealed the presence of 5 genotypes (CA/CG, CG/CG, TG/CA, TG/CG

5 Page 5 of 9 and TG/TG). Presence of TG/TG, TG/CG and CG/CG was correlated with presence of A/A(12/15), A/G(11/15) and G/G(11/15) genotype of rs , suggesting that rs and rs were in linkage disequilibrium with CCR6DNP, which has been shown to correlate with the expression level of CCR6(3). In conclusion, the current data demonstrate a genetic association between CCR6 variants and susceptibility to LN, further demonstrating a potential role of Th17 cells in SLE pathogenesis. However, more widespread replications and functional assays are still needed.

6 Page 6 of 9 References: 1 Amarilyo G, Lourenço EV, Shi FD, La Cava A. IL-17 promotes murine lupus. J Immunol. 2014;193(2): Martin JC, Baeten DL, Josien R. Emerging role of IL-17 and Th17 cells in systemic lupus erythematosus. Clin Immunol. 2014;154(1): Kochi Y, Okada Y, Suzuki A, Ikari K, Terao C, Takahashi A, et al. A regulatory variant in CCR6 is associated with rheumatoid arthritis susceptibility. Nat Genet. 2010;42(6): Stahl EA, Raychaudhuri S, Remmers EF, Xie G, Eyre S, Thomson BP, et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet. 2010;42(6): Brkic Z, Corneth OB, van Helden-Meeuwsen CG, Dolhain RJ, Maria NI, Paulissen SM, et al. T helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus? Arthritis Res Ther. 2014;16(2):R62. 6 Mendez-Reguera A, Perez-Montesinos G, Alcantara-Hernandez M, Martinez-Estrada V, Cazarin-Barrientos JR, Rojas-Espinosa O, et al. Pathogenic CCR6+ dendritic cells in the skin lesions of discoid lupus patients: a role for damage-associated molecular patterns. Eur J Dermatol. 2013;23(2): Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40(9): Zhou XJ, Lu XL, Lv JC, Yang HZ, Qin LX, Zhao MH, et al. Genetic association of PRDM1-ATG5 intergenic region and autophagy with systemic lupus erythematosus in a Chinese population. Ann Rheum Dis. 2011;70(7): Zhou XJ, Nath SK, Qi YY, Cheng FJ, Yang HZ, Zhang Y, et al. Identification of MTMR3 as a novel susceptibility gene for lupus nephritis in northern Han Chinese by shared-gene analysis with IgA nephropathy. Arthritis Rheumatol. 2014;66(10):

7 Page 7 of 9 Table legends: Table 1. Associations between CCR6 variants and susceptibility to lupus nephritis (LN). Table 2. Associations between CCR6 variants and susceptibility to SLE stratified by renal involvement.

8 Page 8 of 9 Table 1. Associations between CCR6 variants and susceptibility to lupus nephritis (LN). Discovery 500 LN /900 Controls Replication 626 LN /1932 Controls Total 1126 LN /2832 Controls SNP Pos (hg19) Minor MAF Allele OR MAF Allele OR MAF case/ Allele OR allele case/ model p (95% CI) case/ model p (95% CI) control model p (95% CI) control control rs A 44.5/ ( ) rs A 44.5/ ( ) 43.2/ ( ) 43.0/ ( ) 43.8/ ( ) 43.6/ ( )

9 Page 9 of 9 Table 2. Associations between CCR6 variants and susceptibility to SLE stratified by renal involvement. SLE vs. Healthy control (2189/2832) LN vs. Healthy control (1126/2832) LN vs. SLE without LN (1126/1063) SNP Genetic model Risk Frequenc p OR (95% CI) Frequency p OR (95% CI) Frequency p OR (95% CI) factor y of risk factor of risk factor of risk factor rs Allele model A 42.2/ ( ) 43.8/ ( ) 43.8/ ( ) Dominant model AA+GA 66.6/ ( ) 68.3/ ( ) 68.3/ ( ) Additive model GA, AA Recessive model AA 17.9/ / / rs Allele model A 42.0/ / / ( ) Dominant model AA+GA 66.5/ ( ) 68.2/ ( ) 68.2/ ( ) Additive model GA, AA Recessive model AA 17.6/ / / Significant associations were marked in bold and OR values were presented for the most fitted genotypic models.